Epidemic typhus, caused by Rickettsia prowazekii, is one of humankind's greatest scourges in the last 500 years. It occurs currently in areas with social disorder and/or poor economic conditions. R. prowazekii is a category B priority potential bioterror pathogen. There is no safe vaccine for preventing epidemic typhus. The long term goal of this project is to understand the pathogenesis of rickettsial diseases and to prevent epidemic typhus. Epidemic typhus can be prevented by vaccination with the attenuated E strain (also called Madrid E strain). However, E strain is not used for vaccination because it causes mild epidemic typhus in 14% of immunized persons, apparently due to reversion to the virulent Evir strain. We hypothesize that a reverse mutation occurred in Evir strain and is the determinant for the virulence of Evir strain and that a more substantial knockout of the mutated gene in Evir strain will attenuate Evir strain and make it a safe vaccine. The objective of this project is to develop an effective and safe vaccine for epidemic typhus and possibly for other rickettsial diseases. Three specific aims are proposed to evaluate our hypothesis and provide a stable live attenuated vaccine. Specific aim 1. Identification of the mutations in the avirulent E strain that revert to wild type in the revertant virulent Evir strain by genomic sequencing;2. Genetic inactivation of the gene(s) with reverse mutation in Evir strain by homologous gene recombination;3. Determining safety, immunogenicity, and protective immunity of the candidate attenuated vaccine in mice, guinea pigs and rhesus monkeys. The knockout strain of Evir strain is expected to be attenuated, and it should be able to stimulate protective immunity in animals like E strain does. However, the gene knockout of Evir strain should be prevented from reversion to the virulent state because the virulence determinant is permanently deleted. Thus, the knockout Evir strain can be a safe and effective vaccine for preventing epidemic typhus. This effective epidemic typhus vaccine may be effective for other rickettsial diseases because of the cross-reactive antigens among the rickettsiae.